Experiment 1: Recovery of ASFV from yard soil on macrophages, a
first pilot experiment
In a pilot experiment, 5 g of yard soil were spiked with 400 µL of
infectious blood at a titer of 7.25 log10HAD50/mL and stored at 4°C or 25°C. In this study, blood
and soil were tested at time points 0, 3, 6, 24, 48, and 72 h. First, 5
ml of RPMI-1640 cell culture medium (Thermo Fisher Scientific, Schwerte,
Germany) with 10% fetal bovine serum (FBS) and 2% antibiotics (Gibco
Penicillin-Streptomycin mix, 10000 U/ml; Thermo Fisher Scientific) was
added to the inoculated soil. Then the soil was agitated in the media by
vortexing for 45 seconds (see Figure 2 for all steps). Next, soil and
media were sonicated for 45 seconds at 4°C with the settings: duty cycle
40%, output 3.5 with a Branson Sonifier 450 (Heinemann Ultraschall- und
Labortechnik; Schwäbisch Gmünd, Germany). After sonication, the soil
suspension was centrifuged for 30 minutes at 2,500 x g at 4°C. The
supernatant was poured over a coffee filter, pushed through a 0.45 µm
syringe filter (Millex Filter Units; Merck Millipore Ltd., Tullagreen,
Ireland) and the filtrate was stored at -80°C prior to titration.